sapstain fungi of some commercially important timbers and their ...

INTRODUCTIONFungi cause different kinds of damages to timber viz. decay, soft rot,sapstain and mould. Mould occurs on the surface of the timber and generally hasa woolly or powdery appearance. Moulds cause superficial staining due to thepresence of mycelium as well as their coloured spore masses. Discolouration ofsapwood caused by fungi is commonly known as sapstain. As the colour thatthese fungi impart to the wood is frequently bluish-gray, the stain is often referredto as blue stain. Stain due to fungi may be superficial or penetrating. Deep seatedpenetrating stains are caused by fungi that have dark coloured hyphae or thatproduce pigments diffusing into the wood tissues. Soon after the tree is felled,the cut ends of the logs and portions from which the bark has been removed, getexposed to infection by spores of staining fungi.Discolouration of wood is one of the defects which causes considerableloss (in quality) of wood products. Sapstain, besides spoiling the appearance ofthe timber, affects the goods stored in contact with the stained wood. In paperindustry, when stained timber is used, more chemicals are required to bleach thepulp. Due to the susceptible nature of some of the timbers to sapstain attack, theyare processed at the earliest to avoid infection and this urgency often causespractical difficulties to the manufacturers.In Kerala, small scale wood-based industries such as packing case, plywoodand match units, utilize large quantity of wood from miscellaneous treespecies. It is estimated that there are about 460 packing case, 81 plywood and 144match units in the State. The major timber species employed in match industriesin Kerala are Bombax ceiba (Linn.), Ailanthus triphysa (Dennst) Alston, Alstoniascholaris (Linn.) R. Br and some other soft woods available. In recent years, withthe dwindling natural forest resources it has become necessary to depend on otheralternative timber sources to meet the current and future needs of wood. Heveabrasiliensis (HBK.) Muell. Arg. (rubber wood) with some positive attributes, meritsconsideration when we look for an alternative and cheap timber. After extractingthe latex, rubber trees are felled for replanting the area and the wood thus obtainedis being utilized now-a-days as the main source of wood for manufacturingpacking-cases.In India, not much work has been done on the effect of sapstain fungi onwood. Some studies on decay and sapstain of timbers, their causes and preventionwere conducted by Bakshi (1953). The cause of discolouration of rosewood(Dalbergia latifolia (Roxb.) veneers was investigated by Ananthanarayanan, (1971).Due to favourable environmental conditions such as high rainfall and humidity,sapstain is a serious problem in Kerala. However, no work has been done onsapstain of commercially important timbers in the State. This was the relevanceand context of the present study, attempting to find out the microorganismsresponsible for sapstain in selected timbers and possibilities of their chemical

control. Due to the commercial importance of rubber wood and its high susceptibilityto fungal attack studies were carried out to find out the sapstain fungiparticularly on this timber. Since Botryodiplodia theobromae Pat. was the dominantfungus causing stain on most of the timbers, studies were also undertaken toinvestigate various aspects of infection by this fungus on rubber wood.

MATERIALS AND METHODSSurveyA preliminary survey was conducted in some of the wood-based industriessuch as plywood, packing case and match factories in Trichur District andeight timber species were selected for the study, viz., Ailanthus triphysa (Dennst.),Alston (Matty), Alstonia scholaris ( Lm.) R. Br. (Ezhilam-pala), Anacardium occiden taleLinn. (Cashew), Bombax ceiba (Linn.) (Mullilavu), Erythrina stricta Roxb. (Murukku),Hevea brasiliensis (HBK.) Muell. Arg. (Rubber), Macaranga peltata (Roxb.) Muell.Arg. (Vatta) and Mangifera indica Linn. (Mango). Wood samples of these specieswere collected at monthly interval from wood based industries in Trichur Districtto isolate the mould and stain fungi growing on them. In addition, samples werealso collected from Calicut, Malappuram, Palghat, Ernakulam, Kottayam, Alleppey,Quilon and Trivandrum Districts, but only once.Isolation of microorganismsA small piece of the stained wood was surface sterilized using 0.1% HgCl 2solution, washed thoroughly in several changes of sterile distilled water andplated on potato dextrose agar (PDA). The plates were incubated at 282 2 O C andobserved for the growth of any fungus. After 5 days, the fungus growing aroundthe wood sample was isolated, purified and maintained on PDA slants for furtherstudies.Identification of isolatesCultural and morphological characteristics of isolates were studied andfor the confirmation of their identity, cultures were referred to the CAB InternationalMycological Institute, Kew, UK. These cultures were periodically subculturedand stored for further investigations.Susceptibility testFungi isolated from various timbers were evaluated for their ability tostain healthy wood. For inoculation, unstained sapwood blocks (7 x 5 x 1 cm)were cut from freshly harvested green timber and steam sterilized for 15 minutes.A disc of 8 mm dia taken from the edge of an actively growing culture of the testfungus was placed over the surface of each block. The ,inoculated blocks were

then placed on'glass rod supports over moistened sterile filter papers in sterilePetri dishes and these set-ups were incubated at 28 + 2°C for 15 days. Eachisolate was tested on 5 replicate blocks; controls were also maintained with plainagar discs. After the incubation period, the inoculated blocks were examined forgrowth of the fungi on the surface and internal stain after splitting them open.Weight loss due to Botryodiplodia theobromaeWeight loss in wood blocks of Ailanthus triphysa, Alstonia scholaris, andHevea brasiliensis (widely used for making match splints/packing cases) due to theinfection by Botryodiplodia theobromae Pat., the most dominant fungus causingsapstain was studied over a period of four months following the procedure describedby Stranks (1976). Forty test blocks (5.5 x 1.5 x 0.5cm size) of each timberwere cut from clear fresh sapwood, oven dried at 105°C for a few days till theweight became constant and their initial dry weight recorded. The sample blockswere then placed over a wet filter paper in a closed damp chamber to bring themback to normal moisture equilibrium. Following steam sterilization for 15 mintwenty blocks were inoculated with 8 mm dia agar disc taken from the edge ofa 5-day-old culture of B. theobromae. These blocks were incubated in large testtubes (14.0 x 25.0 cm) containing 15 ml of sterile water. The atmosphere aroundthe inoculated blocks was humidified by inserting a paper wick (2.5 x 10 cm)covering the entire length of the block. Each tube contained 15 ml of sterile waterin which the lower end of the wick was immersed; the inoculated wood blockswere kept free of direct water contact by glass supports projecting above thewater level. The tubes were closed with cotton plugs (Fig. 1). The remainingblocks were inoculated with plain agar discs and these blocks were used ascontrol. The set-ups were incubated at 28 + 2°C. Every month, five blocks eachfrom inoculated and control sets were taken out from the tubes. After removingthe mycelial growth under running tap water, the blocks were dried at 105°C fora few days and their weight recorded.Effect of wood moisture content and microclimatic factors on the growthof B. theobromaeEffect of varying moisture content (MC) of rubber wood on the growthof Botryodiplodia theobromae was studied at three different temperatures (T) andfive different relative humidity (RH) regimes. Different RH levels were maintainedusing saturated solutions of various salts (O'Brien, 1948). The salt solutionsused for maintaining different relative humidities were prepared in sterilewater and each sterile Horlicks bottle contained 30 ml of solution (Table 1). Formaintaining cent percent humidity, 30 ml of sterile water was used in the bottles.Fresh oven dried rubber wood blocks (3.3 x 4.0 x 2.5 cm) were immersed in waterfor appropriate periods so that they attain approximate MC of 50, 75 and 100%.These wood blocks were then steam sterilized for 15 minutes and inoculated withan 8 mm dia disc.

Table 1 Relative humidities over saturated solutions at various temperatures(after O'Brien, 1948).Salt Relative humidity Temperature 0 CpercentageWater 100 20ZnSO 4 7H 2 O 90 20NH 4 CI 80 20NH 4 NO, 67.4 20Ca(NO 3 ) 2 4H 2 O 56.0 20Water 100 30KNO 3 91.5 30(NH 4 ) 2 SO 4 81 .0 30CO(NH 2 ) 272.9 30NH 4 NO 3 60.0 30Water 100 40KNO 389.4 40KC1 82.0 40NaNO 370.5 40CO(NH 2) 268.7 40taken from an actively growing B. theobromae culture. The wood blocks wereselected randomly and hung inside the bottles above the solution using stainlesssteel wire (Fig 2a). The controls were inoculated with plain agar discs (Fig. 2b).The inoculated and the control wood blocks were incubated for 3 months at threedifferent temperatures viz. 20,30 and 40 0 C. Each treatment had five inoculatedand five control blocks. The growth of fungi over the surface of wood blocks wasexamined visually and assessed using the following rating index (Table 2) afterfour weeks of incubation. The data were subjected to analysis of variance (Snedecorand Cochran, 1967).Table 2 Rating index for assessing the fungal growth (after Plackett, 1982).RatingGrowthNo growthTrace growthLight growthMedium growthHeavy growth5

Figs. 1 - 2. la. Control block inoculated with plain agar disc. lb. Test tubes withrubber wood blocks inoculated with B. theobromae for assessing weight loss.2a. Rubber wood block inoculated with B. theobromae. 2b. Control block inoculatedwith plain agar disc.

Optimum moisture content of wood for the growth of B. theobromaeOptimum moisture content of rubber wood required for maximum growthof B. theobromae was determined. Two hundred wood blocks (7.0 x 5.0 x 1.0 cm)made from freshly sawn rubber wood were kept in an incubator maintained at35°C. Twenty sample blocks were removed each day for 10 consecutive daysfrom the incubator and steam sterilized for 15 minutes. The initial weight of 10sterilized blocks was recorded and blocks were oven dried at 105°C for theestimation of final weight. Of the remaining 10 blocks, five were inoculated with8 mm dia disc of an actively growing culture of B. theobromue and the rest withplain agar disc to serve as control. All the blocks were incubated in sterile Petridishes for 2 weeks at 28 + 2°C. Moisture content of the inoculated blocks wasdetermined by calculating the corresponding oven dry weight of blocks. After theincubation period, the inoculated blocks were observed for the growth of thefungus on the surface. The growth was assessed visually using the rating indexas given under Table 2.Chemical control of sapstain and mould growth(a) Evaluation of various fungicides using sterile wood blocksThe efficacy of some of the commonly available fungicides viz.carbendazim, carboxin, copper oxychloride, mancozeb, sodium azide, thiram andziram were tested at 1% a.i. against stain as well as mould fungi. Further, theefficacy of sodium azide was tested at concentrations of 0.1, 0.25, 0.5 and 0.75 %Since higher concentrations were found to be effective in controlling stain andmould fungi, the chemical was also tested in lower concentrations. (10, 50, 100,250 and 500 ppm). All fungicidal evaluations were done using freshly sawn rubberwood blocks (7.0 x 5.0 x 1.0 cm size) as rubber wood was very susceptible tomould, sapstain and decay fungi (Ali et al., 1980).Sterile wood blocks were dipped in fungicidal solution for 30 seconds andexcess solution drained completely before placing inside the Petri dishes forinoculation. The test wood blocks were inoculated with 8 mm dia disc fromactively growing cultures of Botryodiplodia theobromae, Ceru tocystis fimbriata Ellis &Halstead, Acremoniunr spp., Fusarium sp., Scytalidium lignicola Pesante, Aspergillusniger Van Tieghem, Aspergillus flavus Link ex Gray, Trichoderma viride Pers. exGray, and Memnoniella echinata (Riv.) Galloway. Wood blocks dipped in distilledwater served as control and blocks dipped in solutions containing 0.5% sodiumpentachlorophenoxide (NaPCP) plus 1.5% borax as standard (Butcher, 1980). Foreach fungicidal treatment, there were six replicate wood blocks. The Petri disheswere incubated at 28 + 2°C for 2 weeks. The fungal growth observed on theblocks was visually assessed using the rating index (Table 2) and the percentageinhibition of fungal growth over control calculated.

As captafol had been found to be very effective against stain and decayfungi (Butcher, 1980) three different concentrations (1.0%, 1.5% and 2.0% a.i.) ofthis fungicide were also tested.(b) Evaluation of sodium azide against stain and mould fungi using freshnonsterile wood blocksSince sodium azide has proved to be a promising chemical for the controlof stain and mould fungi even at very low concentrations, some more detailedstudies involving this chemical were attempted. The efficacy of sodium azide toprotect fresh nonsterile rubber wood blocks from fungal growth was studied overa period of one month. Twenty five freshly cut unstained rubber wood blockswere dipped in sodium azide solution of 0.1,0.25,0.5 and 0.75% concentration for30 seconds and kept inside nonsterile Petri dishes moistened with filter papers tomaintain humidity. Fungal growth over the blocks was recorded after four weeks.The percentage of control of fungal growth was calculated by counting infectedand healthy wood blocks.(c) Evaluation of sodium azide against stain and mould fungi using preincubatednonsterile wood blocksThe effect of sodium azide to control mould and stain fungi was testedusing nonsterile preincubated wood blocks. Twenty five freshly cut rubber woodblocks were kept in open air at the saw mill for an hour. These blocks were thentransferred to a humid chamber for 2 days so that the spores of the mould as wellas stain fungi fallen over the blocks grew and established over the wood blocks.These wood blocks were dipped in sodium azide solutions of 0.25,0.5, and 0.75%concentration for 30 seconds and incubated in Petri dishes. Observations weretaken at weekly intervals for one month. The growth of the fungus over theblocks was assessed visually. The efficacy of the chemical was calculated bycounting the number of infected and healthy wood blocks and the data on thepercentage of inhibition of fungal growth at the end of the fourth week tested bystandard normal deviate test.(d) Effect of dipping time on the control of fungal growth over the wood blocksFreshly cut rubber wood blocks were dipped in sodium azide solutionsof 0.1, 0.25, 0.5 % concentration for 10, 20 and 30 minutes. The excess solutionwas drained off from the wood blocks and incubated in Petri dishes for fourweeks. Control blocks were dipped in sterile water for 10, 20 and 30 minutes.Weekly observations on fungal growth over the wood blocks were taken for aperiod of one month. The percentage of control of fungal growth was calculatedby recording the number of infected and noninfected wood blocks and the dataanalysed statistically using standard normal deviate test.

Treatment 1 : Dipping and stacking without spaceThe rubber wood planks were dipped in bacterial suspension for 10 seconds,drained completely to remove excess solution and then piled one above theother. Each pile comprised of four individual planks and for each observationthere were four replicates. Control planks were dipped in water and stacked inthe same manner.Treatment 2 : Dipping and stacking with spaceAfter dipping in bacterial suspension the planks were stacked one abovethe other, but separated from each other by a reeper of size 15 x 6 x 1.0 cm soas to have some air space in between the planks. Controls dipped in water werealso stacked in the same way.Treatment 3 : Spraying and stacking without spaceThe wood planks were uniformly sprayed with the bacterial suspensionusing a sprayer. Controls sprayed with water were also maintained. Planks werethen stacked without any space in between.Treatment 4 : Spraying and stacking with spacePlanks were stacked one above the other after spraying with bacterialsuspension, but separated individually using a reeper. Controls were maintainedby spraying with water.Observations on fungal growth were recorded fortnightly for a period oftwo months. At each observation, samples were taken randomly and outline offungal growth on either side of the planks was marked and drawn on a paper.The area covered by fungal growth as marked on the paper was then determinedusing a 'Licor' leaf area meter. The data obtained at the end of the fourth weekwere subjected to analysis of variance.

RESULTS AND DISCUSSIONThe common stain causing fungi isolated from different timber speciessuch as Hevea brasiliensis, Ailanthus triphysa, Bombax ceiba, Alstonia scholaris, Mangiferaindica, Anacardium occiden tale, Macaranga peltata and Erythrina s tricta wereBotryodiplodia theobrontae, Ceratocystis fimbriata, Acremoniumspp., Scytalidium lignicolaand Fusarium spp. of which B. theobromae was the most dominant on causing stain(Figs. 3, 4). B. theobrontae was reported as the most prevalent sapstain fungus ontropical woods (Cartwright and Findlay, 1958; Olofinboba, 1974; Hong, 1976). Itwas also known to cause sapstain in rubber wood in Malaysia (Ali et al., 1980).

Ceratocystis fimbriata was prominently found causing stain in rubber wood duringrainy season in Kerala. Ceratocystis coerulescens was reported as the most importantstain fungus on hardwoods in the southern USA (Verrall, 1941). In Russia,Cera tocystis spp. were responsible for the discolouration of the wood (Vanin,1932). C. picea, normally a nonstaining fungus on conifers in the western USA,caused stain in red Alder (Alnus rubra) (Morrell, 1987). Several species of Ceratocystiswere reported to cause stain on pine wood in Spain (Troya and Navarrete, 1989).The fungi isolated from the surface of different timber species surveyedwere Aspergillus niger, A. flavus, Penicillium spp., Trichoderma viride, Memnoniellaechinata, Syncephalastrum racemosum Cohn ex Schrot., Cladosporium sp., Mucor sp.,and Absidia corymbifera (Cohn) Sacc. & Trotter, of which Aspergillus spp., Penicilliumspp., T. viride, S. racemosum and Mucor sp., were the dominant ones (Fig. 5).M. echinata was seen more on rubber wood (Fig. 6).The results of the survey indicated that there was no definite pattern inthe occurrence of mould and sapstain on different timber species collected. Alltimber species surveyed were found to be susceptible to B. fheobromae; H. brasiliensisbeing the most severely and frequently affected. Based on the frequency ofisolation of B. theobromae, it can be concluded that timbers such as H. brasiliensis,A. occidentale, Artocarpus spp. and A. triphysa were the most susceptible ones whileM. indica, B. ceiba and E. stricta moderately susceptible; least susceptible timberswere A. scholaris, and M. peltata (Table 3).Table 3.Frequency percentage of Botryodiplodia theobromae in fungalisolations made from various timbers of Kerala.Timber Total No.of Frequency of Number of monthsspecies isolations infection samples collectedpercentage in a yearAilanthus triphysaAlstonia scholarisAnacardium occidentaleArtocarpus spp.Bombax ceibaCeiba pentandraErythrina strictaHevea brasiliensisMacaranga pelta taMangifera indica1311131143756622357018777540426081275776647533149

A survey conducted in various wood industries of Trichur District revealedthat there was no clear pattern of attack of B. theobromae on rubber woodthroughout the year. The incidence was observed to be more than 50% in all themonths of collection (Table 4). Even in drier months cent percent infection wasnoted.Table 4Percentage isolation of Botryodiplodia theobromae on rubber woodfrom various places in Trichur District during the year 1987.Locality Month Percent isolation of B. theobromaeOllurChiyyaramPudukkadAlurOllur011 urKodakaraKalletumkaraOllurChi yyaramMarathakaraJanuaryMarchAprilMayJuneJulyAugustSeptemberOctoberNovemberDecember67100100507010010010010010070Susceptibility testIn the artificial inoculation trial, the test fungus grew from the culture discand spread over the upper side of the wood block within five days. After fifteendays, the whole block was covered by fungal mycelium. When the superficialmycelia were scraped using a scalpel blade, surfaces of the test blocks of differenttimbers were found to be stained. In the case of B. theobromae, the stainingpenetrated to a depth of 4 to 8 mm inside as seen after splitting the blocks. Thecontrol samples remained unstained.Weight loss due to Botryodiplodia theobromaeStudies revealed that there was a weight loss in rubber wood in the firstmonth which increased to 12.2% by the end of fourth month (Table 5). In the caseof Ailanthus triphysa the weight loss increased from 4.3% in the first month to10.1% in the fourth month. In Alstonia scholaris there was no weight loss notedin the first three months, but at the end of fourth month a weight loss of 4.5%occurred. The results clearly showed that rubber wood is the most susceptibleto sapstain caused by B. theobromae while A. scholaris is the least. These resultsalso confirm the findings of the survey.

Table 5Percentage of weight loss due to B. theobromae on different woodspecies for a period of four months.Months of incubation A. triphysa A.scholaris H. brasiliensis12344.3 0.0 8.05.0 0.0 8.39.9 0.0 8.510.1 4.5 12.2The attack of sapstain fungi on the physical and strength properties ofwood has not been studied earlier in detail. Findlay and Pettifor (1939) reportedthat a loss of 30-40% in toughness and 20% in bending strength was noted inobechi (Triplochiton scleroxylon) stained heavily with B. theobromae. Pinheiro (1971)also recorded a decrease in bending strength of poplar wood due to the attack bythe same organism. Umezurike (1969) showed that the blue stain fungus, B.theobroniae was capable of degrading cellulose in wood of Bombax buonopozense P.Beauv., particularly after utilizing starch and soluble carbohydrates. The modeof degradation of wood blocks of Gossweilerodendron balsamiferum(Verm.). Harms.,a tropical forest tree extensively used for construction work in Nigeria, by B.theobromae has been studied under laboratory conditions by Umezurike (1978).He recorded a 5-7% weight loss of infected wood blocks. The pattern of invasionof wood blocks by B. theobromae has been found to be similar to those of soft rotfungi (Krapivina, 1960; Levy, 1967; Umezurike, 1969). As both soft rot and bluestain fungi are capable of forming chains of cavities in the S2 layer of the secondarycell wall of wood, the main difference between these two groups of fungi lies,apparently, in the ability of blue stain fungi to synthesise pigment material bysecondary metabolism. The weight loss of rubber wood and A. triphysa reportedhere may be due to the degradation of cellulose. The susceptibile nature of rubberwood to B. theobromae may be either due to high percentage of starch content inthe tissues or the presence of any chemical which promotes the fungal growth.However, this was not verified. Fougerousse (1985) reported that susceptibility toblue stain depends on the predominance of parenchymatic tissues or the physiologicalcondition like higher percentage of starch in the tree at the time of felling.Effect of wood moisture content and microclimatic factors on the growth of B.theobromaeAt relative humidity (RH) 60,70 and 80% no fungal growth was recordedat 20 0 C (T) even at cent percent moisture content (MC) of timber. But fairly goodgrowth occurred at 100% RH in three moisture content levels. At 30 0 C, profusegrowth of the fungus was noted at 90% and 100% RH; the fungal growth was notmuch influenced by the moisture content of timber. At 40 0 C, in all combinations

of RH and MC, the growth of the fungus was not very much restricted, butcomparatively better growth was noted at 100% RH. It is presumed that thegrowth of the fungus on wood blocks was influenced by higher percentage ofrelative humidity. Statistical analysis showed significant difference between thetreatments (Table 6). Treatments (T, MC, RH), 20" C, 100%, 100%; 30" C, 75%,100% and300C, 100%, 100% were found to be significant as compared to all othertreatments. Hong (1980) studied the temperature tolerance of B. theobromae onagar medium as well as on wood blocks. He found that the optimum temperaturefor growth of B. theobromae on malt agar was very close to 30°C. But on woodblocks the fungus could survive at higher temperatures. In the present study also,30°C was found to be the ideal temperature for good growth of the fungus.Viitanen and Paajanen (1988) studied the growth of mould fungi such asAspergillus versicolor, Cladosporium sphaerospermum, Penicillium sp. and Aureobasidiumpullullans in different RH and temperature. He found that the moulds grewrapidly in higher humidities (RH > 96%) and the lowest humidity for slow growthwas 80%. Generally most of the fungi grow well in higher humidities (95% ormore) (Cochrane, 1958). From the present study, it was clear that when thehumidity was high, the wood blocks having low moisture content, absorbedmoisture from the surroundings and enhanced the growth of B. theobromae onwood blocks.Table 6Growth rate index of B. theobromae grown on rubber woodblocks of different moisture contents (MC) incubated at varioustemperatures and relative humidity (RH).200 300 400Moisture content Moisture content Moisture content50 75 100 50 75 100 50 75 10060 o a *o a O a O a 0 a O a 0 a 0 a 0 a70 0 a O a O a 0.2 a 0.4 a O.8 a 0 a 0 a 0 a80 0 a O a O a O a 1.4 a 1.6 a 0 a 0.2 a 1.6 a90 1.2 b l.8 b 3.3 c 2.8 b 3.4 c 3.0 b 0.2 a 0.4 a 1.2 b100 3.4 c 3.4 c 3.8 c 3.4 c 3.6 c 4.0 C 0.2 a 1.0 a 2.8 b* Values superscribed by the same letter are not statistically differentOptimum moisture content for the growth ofB. theobromaeGrowth rating of B. theobromae on rubber wood at different moisturecontents is given in Table 7. Above 29% moisture content, heavy growth of the

Table 7Growth rating of B.theobromae at different moisture contents (MC)of woodS1.No. MC% Rating*1 65.63 42 46.05 3.83 29.49 44 27.02 2.45 27.04 2.26 25.79 1.47 25.86 1 .08 23.83 0.09 16.18 0.010 15.85 0.0* For explanation see Table 2.fungus was seen on the wood blocks. In 27% moisture content, light growth ofB. theobromae was noted where as in 25% only traces of fungal growth was observed.Below 25% the fungal growth was found to be very restricted. This studyhas confirmed earlier reports on the lowest moisture content of wood essential forthe fungal growth. Pinheiro (1971) studied the maximum and minimum moisturecontents of poplar wood which favoured blue stain attack by B. theobromae. Hefound that the lowest moisture content which permitted the growth of the funguswas 24%. Colley and Rumbold (1930) also found that the lower limit for staindevelopment in loblolly pine by Ceratocystis pilifera was 24%. From this study alsoit was clear that if the moisture content of the wood is reduced to

Table 8Evaluation of fungicides for the efficacy against various staining andmould fungi (Percent inhibition).ChemicalsConcentrationAf An Me Tv Bt Cf Fs As Sl%a.i.Carbendazim 1 00 90 00 00 00 00 85 90 50Sodium azide 1 100 100 100 100 100 100 100 100 100Carboxin 1 00 00 100 00 100 25 100 00 100Thiride 1 00 50 90 00 90 00 50 00 00Copperoxychloride l 00 00 80 00 00 00 00 50 25Ziram 1 00 00 90 00 90 00 00 50 50Mancozeb 1 00 00 100 00 50 00 00 00 00Captafol 1 90 90 100 00 10 00 90 90 100/I"1.5 95 95 100 00 00 00 90 90 1002 95 95 100 10 00 00 90 90 100NaPCP+Borax 0.5+1.5 100 100 100 100 100 100 100 100 100Control 00 00 00 00 00 00 00 00 00 00Af - Aspergillus flavusTv - Trichoderma virideFs - Fusarium solaniAn - Aspergillus nigerBt - Botryodiplodia theobromaeAs - Acremonium strictumMe - Memnoniella echinataCf - Ceratocystis fimbriataSl - Scytalidiuni lignicolaSodium pentachlorophenoxide (NaPCP) is widely used as a preservativefor the control of sapstain fungi, moulds and decay fungi. A concentration of 0.4to 0.5% a.i. is suggested to control sapstain (Anon, 1972). During the last fewyears much work has been carried out to find out a suitable substitute for NaPCPwhich has high mammalian toxicity. In the field tests conducted in Brazil, Busan,a fungicide at 1.5% a.i. proved to be effective against sapstain and mould fungi(Milano, 1981). In the field tests for a period of five months Plackett (1982) provedthat Busan 1.2% a.i. was effective against sapstain and mould fungi. Undervarious trade names, copper-8-quinolinolate has been tested in the laboratory orin the field. In laboratory tests against several sapstain and mould fungi Cassensand Eslyn (1981) obtained rather poor results with 0.02% a.i. on yellow poplar(Liriodendron tulipifera) at 0.02% a.i., the highest tested concentration; while Butcher(1980) found it effective on Pinus radiata against sapstain, mould and decay fungiat 0.025% a.i. concentration.Many studies have been reported on the efficacy of preservatives for thecontrol of B. theobromae causing sapstain (Tan et al., 1980; Hong, 1981). Ali et al.,(1980) have screened 11 fungicides against B. theobromae, Aspergillus sp., and Penicilliumsp., on rubber wood. They found that captafol was effective to all the threefungi at 1-2% a.i. Gnanaharan (1983) made tests on rubber wood boards treated

y the boron diffusion process and found that 0.5% NaPCP was effective againstsapstain and moulds. Butcher and Drysdale (1974) screened various chemicalsagainst sapstain in New Zealand and reported that captafol 0.2 to 0.3% a.i. wassuperior to NaPCP. Later, Butcher (1980) found that 0.15% captafol was adequatefor long-term protection of pine timber. But in the present study captafol evenat 2% was not at all effective against B. theobromae in the laboratory screening.Evaluation of sodium azide at 0.1, 0.25, 0.5 and 0.75% revealed that thechemical was 100% effective at all concentrations tested on sterile wood blocks.When further tested the chemical was effective at 250 and 500 pprn only (Table 9).b) Evaluation of sodium azide against stain and mould fungi using freshnonsterile and preincubated wood blocks.The results indicated that 80% control of fungal growth was noted at theend of the 4th week in fresh nonsterile wood blocks dipped in sodium azidesolution of 0.75%, whereas in preincubated wood blocks only 60% inhibition offungal growth was noted (Table 10). These two treatments were found statisticallysignificant from all other treatments. It is presumed that, in preincubatedwood blocks some fungi which might have established over the wood blocks priorto the treatment, continued to grow during the incubation period even after thetreatment. But in fresh nonsterile ones, since the dipping was done immediatelyafter cutting, the fungi might not have got favourable conditions or chance toestablish. From this it is clear that a prophylatic treatment immediately afterfelling or cutting will always be better rather than controlling the stain after theinfection has occurred.Table 9Efficacy of sodium azide against staining and mould fungi atdifferent concentrations (Percentage of inhibition).Sodium azideconcentration Af An Me Tv Bt Cf Fs As Sl10 ppm 00 00 00 00 00 00 00 00 0050 ppm 00 00 00 00 00 00 00 00 0100 ppm 00. 00 00 00 00 00 00 00 00250 ppm 85 100 100 100 90 100 100 100 100500 ppm 100 100 100 100 100 100 100 100 100Af - Aspergillus flavusTv - Trichoderma virideFs - Fusarium solaniAn - Aspergillus niger Me - Memnoniella echinataBt - Botryodiplodia theobromae Cf - CeratocystisfinibriataAs - Acremonium strictum Sl - Scytalidium lignicola

~~~~~ ~~~ ~ ~Table 10 Percent control of fungal growth on unsterile and preincubated woodblocks dipped in sodium azide solution.Period of incubation (weeks)Concentration % Is t 2nd 3rd 4th0.25 32 32 32 28Fresh nonsterile 0.5 32 32 32 320.75 88 88 80 80control 0 0 0 00.25 28 12 12 12Preincubated 0.5 56 48 28 280.75 68 64 60 60control 0 0 0 0c) Effect of dipping time on the control of fungal growth over the wood blocksIn the wood blocks dipped in 0.5% sodium azide for 30 minutes, 100%inhibition was noted at the end of the fourth week whereas in 10 and 20 minutesdipping, inhibition was 60 and 92% respectively (Table 11). Two treatments, suchas dipping in 0.5% concentration of sodium azide for 20 or 30 minutes were foundsignificantly different from all other treatments. When the dipping time wasincreased, the percentage of inhibition of fungal growth also increased. Thisincrease in inhibition of growth may be due to the diffusion of more chemicalsolution during long dipping periods.The use of sodium azide as a preservative chemical against mould andsapstain fungi has not been reported earlier. However, it has been used effectivelyto control a few fungal diseases. Row et al., (1974) reported thatCylindrocladium black rot of peanuts was effectively controlled by applying sodiumazide in the soil. Similarly 10 ppm solution of potassium azide inhibitedthe germination of microsclerotia of Cylindrocladium floridanum (Weaver, 1971).Sodium azide was found to be effective in controlling the nursery diseases ofeucalypts (Sharma and Mohanan, 1991). They have found that in laboratoryevaluation, sodium azide (0.05 and 0.1%) was 100% effective against Cylindrocladiumquinqueseptatum, C. ilicicola, C. floridanum, C. parvum and C. camelliae. All thesereports indicate that sodium azide is an effective chemical for controlling fungalgrowth. However, only after studying the toxicity and testing the efficacy in thefield, it can be recommended as a preservative against stain and mould fungi.

Table 11 Percent control of fungal growth on wood blocks dipped in sodiumazide solution for 10, 20 and 30 minutes.Period of incubation (weeks)Dipping time Concentration 1st 2nd 3rd 4th(minutes) %10 0.1 100 100 80 400.25 100 100 100 480.5 100 100 100 60control 0 0 0 0200.1 100 100 70 600.25 100 80 80 800.5 100 100 100 92control 0 0 0 030 0.1 100 100 72 600.25 100 100 100 800.5 100 100 100 100control 0 0 0 0Biological control(a) Testing of the bacterium on culture mediumThe bacterium, Bucilliis subtilis inhibited the growth of all fungi testedexcept Pythium sp. (Table 12). The maximum inhibitory zone was noted in thecase of B. theobromae (Figs. 7, 8) followed by Memnoniella echinata, Scytalidiumlignicola, Trichoderttia viride, Acremonium recefei, Aspergillus niger, A. flavus andCerutocystis fimbriata Among the pathogens tested, Pestulotiopsis sp., the commonfoliar pathogen had shown the inhibitory zone upto 17 mm.Testing of the bacterium on wood blocksThe wood blocks of three timber species, Hevea brasiliensis, Ailanthus triphysAand Alstonia scholaris treated with the suspension of B. subtilis were found to bedevoid of any fungal growth (Figs. 9,l0). On the control blocks the fungus grewwell and covered the entire surface of wood and also resulted in internal stain.

016

-Table 12 Inhibitory zone (in mm) produced by antagonisticagainst various fungi.Bacillus subtilisNo. Fungi tested Clear zone1 Aspergillus flavus 62 A. niger 73 Trichoderma viride 94 Meninoniella echinata135 Botryodiplodia theobromae186 Acremoniuni recefei97 Scytalidium lignicola118 Cera tocys tis fim bria ta59 Fusarium solani1410 Corticium salnionicolor1411 Sclerotium rolfsii912 Pestalotiopsis sp.1713 Rhizoctonia solani1414 Collectotrichum gloeosporioides1015 Pythium sp.00(c) Field testing of the bacteriumThe maximum control of fungal growth was recorded on rubber woodplanks sprayed with bacterial suspension and piled one above the other withoutleaving any space in between. The analysis of variance showed significant differencebetween treatments at p = 0.01 (Table 13). Treatment 3 was found to besignificant when compared to all other treatments.Table 13 Analysis of variance of data on inhibition of fungal growth onrubber wood planks treated with Bacillus sub tilis suspension.--- -_I--Sources ss DF MSS FTreatments 639443.180 7 91349.026 8.4221**Replication 28163.804 3 9387.935 0.8655( ns)Residual 227772.598 21 10846.314Total 895379.582 31______---__-----_I_________________II___---------------------------------------------------------** Significant at p = 0.01 ns = non-significant-

Biological control of plant pathogens, free from hazards, is a difficult butimportant necessity. The antagonistic potential of certain bacteria can be quiteeffective and if well regulated, provide an industrially useful method for woodprotection. Among the various bacterial antagonists tested Bacillus subtilis hasbeen reported to control several plant pathogens (Dunleavy; 1955; Aldrich andBaker, 1970; Broadbent et al., 1971; Swinburne and Brown, 1976; Singh and Deverall,1984). Results of this study are in agreement with earlier reports on the efficacyof Bacillus subtilis in controlling sapstain and mould on various timber species.Laboratory studies were successfully carried out to evaluate the efficacy ofbacteria as a biological control agent against 5 brown rot and 3 white rot fungi(Benko and Highley, 1990). Tests were also carried out in Australia to screenbacteria effective against fungi causing blue stain (Benko, 1988). Bacillus subtilisstrain 186 inhibited the growth of three sapwood inhabiting fungi on agar andprevented colonisation of spruce blocks placed on agar plates (Bernier et al.,1986). More detailed studies with the same strain of bacterium revealed that itcan be used as a potential biological control agent for sapstain and mould growthon unseasoned timber (Seifert et al., 1987). All these reports indicate that B.subtilis can be used in the biological control of sapstain. However, large scale pilottrials are necessary to work out the economic feasibility of this method.

CONCLUSIONS1. Botryodiplodia theobromae is the predominant sapstain fungus of economicallyimportant timbers of Kerala such as Hevea brasiliensis (Rubber), Ailan thustriphysa (Matty), Bombax ceiba (Elavu), Alstonia scholaris (Ezhilam-pala), Mangiferaindica (Mango), Anacardium occidentale (Cashew), Macaranga peltata (Vatta) andErythrina stricta (Murukku).2. Sapstain caused by B. theobromae effects weight loss in timbers. Weight lossin rubber wood after a period of four months was 12.2%.3. If the wood moisture content is reduced to

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